The development of epithelial cancers in multiple tissues, including the skin, is characterized by abnormalities that are both intrinsic to the cell as well as outside the cell in the extracellular environment. During the most recent funding cycle of this Merit Review, we characterized the mutational spectrum of cutaneous squamous cell carcinoma (SCC), the second most common cancer in the country, long- associated with sunlight exposure and widespread impacts on the U.S. veteran population. Deep sequencing of 100 SCCs and 100 patient-matched normal skin specimens identified highly recurrent mutations in the COL11A1 gene (52% of SCCs), which encodes a secreted collagen chain involved in epithelial-stromal interactions as well as novel ultraviolet (UV)-signature mutations in the intracellular, kinetochore gene, KNSTRN (19% of SCCs). A major role for mutant extracellular collagen chains and mutant kinetochore proteins has not been widely appreciated in cancer before and their presence in SCC may shed light on fundamental cancer processes. This competing renewal will therefore characterize the role of these genes in epidermal tumor progression. First, we will determine the function of recurrently mutated collagen genes in epidermal tumorigenesis. We found collagen chain mutations, concentrated at glycine or proline substitutions in the Gly-X-Y motif (with X and Y commonly proline) alpha helical collagen sequence, in 70% of SCCs, with mutation of COL11A1 the most common. In inherited monogenic human disorders, such as dystrophic epidermolysis bullosa (DEB), comparable mutations in COL7A1 produce dominant-negative collagens that disrupt epithelial-stromal cohesion, suggesting a model in which tumors secrete mutant collagens to induce neoplasia-enabling abnormalities in the surrounding stroma. To explore this possibility, we will generate human epidermal tissue with defined mutant collagens in vivo then examine the resulting functional impact on epidermal invasion and tumorigenesis. Aim I will thus test a model in which secreted mutant collagens disrupt native extracellular matrix to facilitate neoplastic progression. Second, we will define the identity and function of Kinastrin-interacting proteins as well as characterize recurrently mutated kinetochore genes in epidermal tumor progression. We observed that the KinastrinS24F UV-signature hotspot mutant prevalent in SCC accelerates epidermal tumorigenesis in vivo. In doing so, it exerts impacts characteristic of cancer, specifically increased cell division and genomic instability. These impacts are distinct and separable, however, suggesting separate mechanisms of action for each effect. To explore this, we will identify the protein interactome of both wild-type and UV-hotspot mutant KinastrinS24F proteins by vicinal protein labeling and mass spectrometry. Kinastrin-interacting proteins will then be functionally disrupted and the resulting impacts on KinastrinS24F-enhanced tumorigenesis will be determined. Also, given that 77% of SCCs were found to have mutations in additional core kinetochore genes, we also plan to disrupt selected kinetochore genes and study their impact on epidermal tumorigenesis. Aim II will thus test a model in which mutant Kinastrin interacts with discrete sets of proteins to enhance tumor progression and in which mutations in specific other kinetochore genes enable carcinogenesis. At the end of proposed funding, we plan to have gained further insight into mechanisms of epidermal tumor progression as a foundation for future strategies for cancer prevention and treatment.